Padi6 expression patterns in buffalo oocytes and preimplantation embryos.

The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (Padi6), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of Padi6 gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of Padi6 gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of Padi6 transcripts in somatic tissues. Notably, the expression levels of Padi6 in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of Padi6 in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo.

Non-targeted Metabolomics Reveals Metabolic Characteristics of Porcine Atretic Follicles.

Follicular atresia is one of the main factors limiting the reproductive power of domestic animals. At present, the molecular mechanisms involved in porcine follicular atresia at the metabolic level remain unclear. In this study, we divided the follicles of Bama Xiang pigs into healthy follicles (HFs) and atretic follicles (AFs) based on the follicle morphology. The expression of genes related to atresia in granulosa cells (GCs) and the concentration of hormones in the follicular fluid (FF) from HFs and AFs were detected. We then used liquid chromatography-mass spectrometry-based non-targeted metabolomic approach to analyze the metabolites in the FF from HFs and AFs. The results showed that the content of estradiol was significantly lower in AFs than in HFs, whereas that of progesterone was significantly higher in AFs than that in HFs. The expression of BCL2, VEGFA, and CYP19A1 was significantly higher in HFs than in AFs. In contrast, the expression of BAX and CASPASE3 was significantly lower in HFs. A total of 18 differential metabolites (DMs) were identified, including phospholipids, bioactive substances, and amino acids. The DMs were involved in 12 metabolic pathways, including arginine biosynthesis and primary bile acid biosynthesis. The levels of eight DMs were higher in the HF group than those in the AF group (p < 0.01), and those of 10 DMs were higher in the AF group than those in the HF group (p < 0.01). These findings indicate that the metabolic characteristics of porcine AFs are lower levels of lipids such as phospholipids and higher levels of amino acids and bile acids than those in HFs. Disorders of amino acid metabolism and cholic acid metabolism may contribute to porcine follicular atresia.

Integration of transcriptomics and non-targeted metabolomics reveals the underlying mechanism of follicular atresia in Chinese buffalo.

Follicular atresia is a complex physiological process, which results in the waste of follicles and oocytes from the ovary. Elucidating the physiological mechanism of follicular atresia will hopefully reverse the fate of follicles, thereby improve the reproductive efficiency of female animals. However, there are still many gaps to be filled during the follicular atresia process. In this study, we first comprehensively summarized and compared a variety of methods to classify Chinese buffalo follicles with different extent of atresia. Then follicular fluid and granulosa cells from the corresponding follicles with different extent of atresia were collected for non-targeted metabolomics and transcriptomics analysis, respectively. After the detection and analysis of 129 follicles, a reasonable classification standard was formed: on the basis of morphological classification, the relative concentrations of estradiol (E2) and progesterone (PROG) in the follicular fluid were determined, follicles with an estradiol-to-progesterone (E2/PROG) ratio >5 were classified as healthy follicles (HF), 1≤ E2/PROG ≤5 as early atretic follicles (EF) and E2/PROG <1 as late atretic follicles (LF). Correspondingly, follicles with granulosa cells apoptosis rate less than 15 % were divided into HF, 15%-25% were classified as EF and more than 25 % were classified as LF. The integration analysis of non-targeted metabolomics and transcriptomics highlights the following three aspects: (1) Atresia seriously damaged the lipid metabolism homeostasis of follicle, in which PPARγ play important roles. (2) Energy metabolism and nucleotide metabolism of atretic follicles were inhibited. (3) Bilirubin is involved in follicular atresia, and it may be the main force to prevent lipid peroxidation in follicular cells. In summary, results of this study provide new understanding of the molecular mechanisms of Chinese buffalo follicular atresia.

Isolation, characterization, and SREBP1 functional analysis of mammary epithelial cell in buffalo.

Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. Sterol regulatory element-binding protein 1 (SREBP1), an important transcription factor, regulates the expression and activity of enzyme and protein involved in milk fat synthesis to influence on the synthesis and secretion of triglyceride in mammary epithelial cells. In the present study, we successfully isolated buffalo mammary epithelial cell by using enzymatic digestion, and then described the growth characteristics and expression characteristics of mammary epithelial cells. Moreover, we cloned the SREBP1 gene from total RNA isolated from milk fat globule and analyzed the function of the SREBP1 gene. After infected with shRNA-SREBP1 lentiviral particle and treated with fatty acid, the expression trend of ACACA, FABP3, FAS, SCD, ERK1, ERK2, PPARy, and Insigl genes was consistent with the expression trend of SREBP1 gene. These results suggested that SREBP1 gene is a central transcription factor in regulating milk fat synthesis and SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene. The current study will provide a theoretical basis for further reveal the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells. PRACTICAL APPLICATIONS: This study aim to separate and analysis characterization of mammary epithelial cell in buffalo. Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. This study will provide a theoretical basis for further research on the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells.

Ruminal microbiota composition associated with ruminal fermentation parameters and milk yield in lactating buffalo in Guangxi, China-A preliminary study.

The ruminal microbiota of 15 dairy buffalo was characterized using high-throughput 16S rRNA gene amplicon sequencing. Results showed that Bacteroidetes was the dominant bacterial phylum in all rumen samples, followed by Firmicutes, Proteobacteria, Tenericutes and Verrucomicrobia. Butyrivibrio was positively correlated with average milk fat yield (R = 0.55; p = 0.03), average milk total solid yield (R = 0.56; p = 0.03) and standard milk yield (R = 0.52; p = 0.05). Acinetobacter were positively correlated with average milk protein yield (R = 0.56; p = 0.03), average milk total solid yield (R = 0.60; p = 0.02) and standard milk yield (R = 0.57; p = 0.03). Acetobacter was positively correlated with acetate (R = 0.63; p = 0.01), propionate content (R = 0.55; p = 0.03), butyrate content (R = 0.61; p = 0.02) and total VFA (R = 0.62; p = 0.01). The phyla Proteobacteria (R = 0.53; p = 0.04) and genus Prevotella (R = 0.52; p = 0.05) were positively correlated with butyrate content. Correlation analysis suggested that increased Butyrivibrio and Acinetobacter residing in the buffalo rumen could improve milk performance.

Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo.

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection.

BACKGROUND: Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. RESULTS: First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/µL DNA was significantly higher than from those injected with 5 µg/mL. However, 50 ng/µL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/µL DNA. Second, zygotes injected early (7-8 h post-insemination [hpi]) developed better than those injected at mid (12-13 hpi) or late (18-19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. CONCLUSIONS: Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

Evaluation of fasting metabolism of growing water buffalo (Bubalus, Bubalis).

The objectives of the present study were to evaluate fasting metabolism (FM) of water buffalo (Bubalus, Bubalis) at three stages of growth (12, 18 and 24 months) in Guangxi, China. Five female water buffalo were used for each age group and their live weight was on average 254, 326 and 338 kg, respectively. All animals were of average body condition, healthy and de-wormed before start of the study. Prior to a 6-day fasting period, buffalo were offered a mixed diet of forage and concentrates (70% and 30%, dry matter basis) on a restricted nutritional level (419 kJ/kg(0.75) of metabolizable energy, ME) for 15 days. Gas exchanges for each animal were determined for 3 days from day 4 of starvation, using open-circuit respiratory head hoods. Fasting body weight was 0.918 of live weight (P < 0.001, r(2) = 0.99). Both fasting heat production (FHP) and FM (MJ/day) increased significantly with increased age of animals (P < 0.05). Linear regression analysis indicated a positive relationship between fasting body weight (kg(0.75)) and FHP (MJ/day, P < 0.01, r(2) = 0.49) or FM (MJ/day P < 0.01, r(2) = 0.52) when using individual animal data across three groups. However, when expressed as kJ/kg(0.75) of fasting body weight, the differences in FHP or FM between three groups of animals were not significant. The present average FHP and FM (322 and 347 kJ/kg(0.75) of fasting body weight) were compatible to those published in the literature for water buffalo, beef and dairy cattle. The present FM data were also used to estimate net energy (NE(m)) and ME (ME(m)) requirements for maintenance for water buffalo. The results for these two parameters were similar to those for FHP and FM. There was no significant difference between three groups of buffalo in NE(m) or ME(m) when expressed as kJ/kg(0.75) of live weight. The present average NE(m) and ME(m) values (347 and 506 kJ/kg(0.75) of live weight) are close to those proposed by the Agricultural and Food Research Council adopted in UK for beef and dairy cattle. The results indicate that the present FM data can be used as a basis for rationing water buffalo in China.